Snuff dipping cause of the oral cavity pharynx. It is also associated with an increased risk for cancer of the esophagus, pancreas, and urinary bladder. In the past 17 years, oral snuff consumption in the U.S. rose by 61% while use of all other tobacco products significantly declined even in the face of a recent resurgence in cigar smoking. The increase in oral snuff sales is primarily attributed to young white men among whom this form of tobacco use is most popular (up to 20% of white high school students). Incidence rates of cancer of the mouth, tongue, and pharynx have risen alarmingly among white males aged 15-34 years. Ninety percent of the snuff products on the US market have weakly alkaline pH(7.5-8.2) and are therefore causing lesions in oral surfaces. This type of snuff contains relatively high levels of unprotonated nicotine, and nitrite as precursors for carcinogenic tobacco-specific nitrosamines (TSNA) which are also present at high concentration and continued o be formed during snuff dipping. The second type of snuff has smaller market, share is weakly acidic (pH 5.4-5.7), is very low in nitrite, contains protonated nicotine rather than the free base, and also has significantly lower TSNA concentrations. Historically, these brands are "starter brands" from which youngsters soon switch to the stronger, slightly alkaline snuff brands. There is evidence indicating that the levels of carcinogenic TSNA in snuff products can be controlled. The proposed research is to document the relative genotoxicity of both types of snuff in short-term and long-term lip canal bioassays for oral carcinogenicity The short-term assay involves instillation of snuff into the surgically created lip canals of rats for ten weeks and then evaluating TSNA adducts formed with DNA in the oral epithelium. The degree and type of adduct formation will signify genotoxic potential of the snuff products. The well established long-term assay requires snuff instillation for 2 years and will record occurrence of benign and malignant tumors in the mouth, tongue, oral pharynx, as well as in target organs that are distant from the point of application, such as lung and esophagus. Because of high levels of precursors in oral snuff, endogenous formation of carcinogenic TSNA during snuff dipping will as yet elevate the carcinogen burden for the user. We plan to demonstrate the occurrence of endogenous TSNA formation from [5-2H] nicotine and from nornicotine that will be added to each of the two snuff types and given to young men who are habitual snuff dippers. GC-MS analysis of their saliva and urine for NNK metabolites NNAL and NNAL-glucuronides will be facilitated by super-critical fluid extraction as part of a method developed at our institute. We will also assess levels of endogenously formed 8-hydroxydeoxy guanine as a measure of oxidative damage by comparative analyses of urine from snuff dippers, smokers of filter and non-filter cigarettes, and certified non tobacco users.